Most circulating testosterone is tightly bound to sex hormone-binding globulin (SHBG) or weakly bound to albumin. The free and albumin-bound forms comprise about 35% of total testosterone and are believed to be biologically active. Factors that affect SHBG or albumin concentrations, such as hyperthyroidism and posture, therefore result in changes in the bioavailable fraction of testosterone. The isoform and glycosylation status of SHBG also potentially impacts bioavailable testosterone by affecting the dissociation constant (KD) between testosterone and this binding protein.

What testosterone assays are available?

Isotope dilution mass spectrometry (MS) is the reference method for total testosterone measurement, but most labs rely on direct chemiluminescent immunoassays that displace bound forms of testosterone from SHBG and albumin. Results of these immunoassays depend on the effectiveness of displacement. They also suffer from limitations such as lack of specificity for cross-reacting steroids, insufficient analytical sensitivity, and imprecision at low concentrations. Other methods used in clinical labs include liquid chromatography-tandem MS (LC-MS/MS) and gas chromatography MS. Both MS-based assays and immunoassays face the challenges of standardization across platforms and appropriate reference intervals partitioned for gender and age groups.

Free testosterone can be quantified after physical separation from the protein-bound forms, which is achieved through equilibrium dialysis or ultracentrifugation. Equilibrium dialysis is considered the gold standard, although ultracentrifugation has gained popularity, too, due to its significantly shorter turnaround time. With equilibrium dialysis, testosterone isotopes can be introduced into the patient's sample at an exponentially lower concentration than endogenous testosterone. Concentration of free testosterone is then estimated based on total testosterone concentration and the distribution of testosterone isotopes across the dialysis membrane. This method circumvents the need for quantifying extremely low levels of testosterone, as well as potential disturbances to the association equilibrium.

Labs can measure bioavailable testosterone as well by using ammonium sulfate to selectively precipitate the SHBG-bound forms. Also available are radioimmunoassays for free testosterone, which are based on competitive binding of a non-testosterone analog. Their use is not recommended for clinical purposes, though.

All of these free or bioavailable testosterone methods are labor-intensive. As an alternative approach, labs can instead perform calculations based on total testosterone, SHBG, and KD between testosterone and SHBG or albumin. Multiple equations have been derived and published by Sodergard et al., Vermeulen et al., Nanjee et al., Sartorius et al., and Ly et al.

How should labs choose the best method for testosterone measurement?

The answer depends on the lab’s clinical needs, instrument and personnel availability, and financial condition. With regards to analytical performance, assays that are traceable to internationally recognized standardized reference materials are recommended. The College of American Pathologists proficiency surveys provide data on the overall agreement of assays. For methods with substantial variability across labs (e.g., the calculation of free or bioavailable testosterone), labs must perform intensive internal validations before assay implementation.

The Centers for Disease Control and Prevention also maintains a standardization program to certify a network of labs using LC-MS/MS methods that produce results with <6% deviation from the reference method. This program serves as a benchmark against which hospital and reference labs can evaluate their testosterone assay performance.

For clinical practice, the Canadian Men’s Health Foundation recommends a morning total testosterone measurement as the screening for deficiency, to be followed by confirmatory bioavailable or free testosterone tests in patients with equivocally low total testosterone levels. For female patients, the Endocrine Society recognizes elevations in total, free, or bioavailable testosterone as indications of biochemical androgen excess.

Jing Cao is an assistant professor of pathology and immunology at Baylor College of Medicine and associate director of clinical chemistry at Texas Children’s Hospital in Houston. +Email: jxcao@texaschildrens.org